The current typical production processes for microorganisms and their byproducts involve culturing the microbes in flasks, vessels or tanks with suitable media in a batch type process. The cultures are then typically removed from the culture vessels and the desired cells or cell byproducts isolated using a variety of separation techniques. Typically the cell cultures are lost in the extraction process. Production of another batch requires substantial amounts of time for cell growth and population development prior to production of sufficient quantities of cell byproducts so as make the processes economic.
In some processes the desired cell product is maintained within the cell and accordingly cell lysis is necessary. In many other biological processes the desired products are excreted from the cells without the need for cell lysis. Continuous production techniques capable of culturing the cells and removing the desired products thus would provide greater production capability by eliminating the need to recreate a cell population in each batch.
Problems associated with the continuous extraction of cell products have been the desire to maintain high cell concentrations and provide gentle agitation of the cell suspensions to create good mass transfer to and from the cells for nutrition and product removal. These problems have not been fully solved using prior art techniques.
A further problem of prior art cell culturing procedures is the relative inability to accurately determine the defects of various production parameters on production rates. Production parameters such as reactor cell density, nutrient flow, agitation, possible inhibiting or enhancing affects due to high densities of cells or other parameters have been difficult to quantitatively determine using batch processing because of the numerous batches which must be produced and inherent variations in the populations and the resultant statistical variations attendant therewith. The addition of various cell culturing additives has also been hampered because of nonuniform concentrations in the culture due to reactions with cell products or metabolic reaction by the cells which may vary significantly from culture to culture.
In an attempt to procure kinetic information and improve productivities by providing higher cell concentrations many approaches have been developed, all with less than satisfactory results. For example, membrane filtration has been attempted, but it has two major drawbacks: large pressure drop which restricts substrate flow; and frequent clogging requiring cleaning or replacement of the membranes. Immobilization of cells in gels or on a solid supporting matrix such as a hollow fiber can be used to obtain high cell concentrations but these systems suffer from other disadvantages. One is that of limited heat and mass transfer to and from the cells. Another is the alteration of cell function through the process of immobilization on a solid support. With hollow fibers, damage to the integrity of fiber walls often occurs due to pressures exerted by the expanding cell mass. Fluidized bed tower reactors, although they eliminate the need for moving parts and have low capital costs, are limited to use with flocculating systems. Cell recycle also has been used but has a disadvantage in that cells are removed from the reactor for a period of time. This may cause cells to go into a stationary phase of growth which may take some time to reverse upon re-entrance to the fermentor. Several processes which use high cell densities combine reaction and liquid extraction of products in one unit. Shortcomings of these procedures are that the extraction solvent often has an inhibition effect on cell growth and multiplicaton. None of the above procedures offer a good technique to quantify the effects of cell crowding on intrinsic growth kinetics. It is felt that growth-limiting factors may regulate maximum cell concentrations, but presently these cell inhibition effects are not well understood.
In the development of kinetic models to describe cell mediated reaction processes, the rate of cell growth is important in order to describe both the rates of substrate utilization and of product formation. Since kinetic information concerning the very recent development of mammalian culture work is quite limited the following discussion will focus on yeast fermentation kinetics. It is expected that kinetic relationships for the animal cells in this research will taken on similar forms as those for fermentation. The intent of the current project will be to elaborate the most important parameters in determining mammalian cell growth rates.
In commercial fermentation processes it is the exponential cell growth rate, shown by the equation below, which is most important: ##EQU1## The specific growth rate constant, u, in it's simplest form can be written as a function of the substrate concentration, S, and saturation constant, K.sub.s, using the Monod equation: EQU u=u.sub.o S/(K.sub.s +S)
where u.sub.o =maximum specific cell growth rate Although the Monod equation is an over-simplification the model often adequately describes fermentation kinetics in low product or cell inhibition environments.
As product and cell concentrations are increased, concentrations are reached which causes inhibition effects. Product inhibition effects have been well characterized for many systems. However, until the recent emphasis on reactors with high cell concentrations, the inhibitory effects of cell crowding on intrinsic kinetics have not been of great importance. Inhibition may result from cell surface properties causing the transmission of growth inhibiting factors. A modified expression for the specific cell growth rate, which includes product and cell inhibition terms, appears below: ##EQU2##
Most models use a linear function to portray the relationship between specific growth rate and cell concentration. However, linear models have been shown to work well only at lower cell concentrations (5-10 g/l). At higher concentrations these are inaccurate and the exponential relationship for cell growth shown in equation (3), is more effective. An alternative form of the product inhibition term has been used by Aiba, et al. and Boulton, but it loses validity at higher product concentrations.
Substrate and product inhibition effects have been well studied for fermentation systems. However, the maximum cell concentration, X.sub.m, and exponential power factor, m, in the cell inhibition term of equation (3) have yet to receive adequate attention. The lack of previous interest in the cell inhibition term is due, in part, to the fact that many conventional cell reaction processes never reach cell concentrations where the cell inhibition term becomes important. For instance, in batch fermentation processes cell concentrations never reach high values because initial substrate concentrations are kept low to prevent substrate inhibition. In conventional continuous fermentation and mammalian cell cultivation processes a portion of the cell suspensions may be lost due to the removal of reaction products. Recent efforts have been made to improve cell mediated reaction productivities by increasing cell concentrations in biological reactors. The following is a brief discussion of the current literature with regard to increased cell concentrations in: (1) fermentations systems; and (2) mammalian culture systems.